This project will deal with a physicochemical investigation and comparison of the properties of the cytoplasmic and mitochondrial forms of the enzyme malate dehydrogenase isolated from porcine heart. Recent work by this laboratory has implicated the essentiality of four residues in the active center of the mitochondrial enzyme i.e., histidine, cysteine, lysine and arginine. In addition, the work by this laboratory has implicated the essentiality of an arginine and lysine residue in the active center of the cytoplasmic form of the enzyme. Sequence studies are being pursued on labeled peptides containing these residues in order to identify other residues in their proximity. Bifunctional reagents will be utilized in order to investigate the proximity of each of these active center residues to one another. The objective of this work will be to attempt to understand the function of each of these residues in the mechanism of action of these enzymes. Recent work from this laboratory has described the pH and concentration dependent dissociation of the subunits of the mitochondrial enzyme. The relationship of specific amino acid residues to this dissociation phenomenon will be investigated. In addition, studies have been instituted in order to establish the identity of specific amino acid residue which appear to play an essential role in the anticooperative binding of NADH to the active center of this enzyme. Finally an investigation will be undertaken in an attempt to relate the changes in subunit structure to the process of enzymatic activity.